Technetium-99m labeled somatostatin-derived peptides for imaging and therapeutic uses

ABSTRACT

This invention relates to therapeutic reagents and peptides, radiodiagnostic reagents and peptides, and methods for producing labeled radiodiagnostic agents. Specifically, the invention relates to peptide derivatives and analogs of somatostatin, and embodiments of such peptides labeled with technetium-99m (Tc-99m), as well as methods and kits for making, radiolabeling and using such peptides to image sites in a mammalian body. The invention also relates to peptide derivatives and analogues of somatostatin labeled with rhemium-186 ( 186  Re) and rhenium-188 ( 188  Re), and methods and kits for making, radiolabeling and using such peptides therapeutically in a mammalian body.

This application is a divisional of U.S. application Ser. No.07/902,935, filed Jun. 23, 1992 and now U.S. Pat. No. 5,716,596, issuedFeb. 10, 1998.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention relates to therapeutic reagents and peptides,radiodiagnostic reagents and peptides, and methods for producing labeledradiodiagnostic and radiotherapeutic agents. Specifically, the inventionrelates to peptide derivatives and analogues of somatostatin, andembodiments of such peptides labeled with technetium-99m (Tc-99m), aswell as methods and kits for making, radiolabeling and using suchpeptides to image sites in a mammalian body. The invention also relatesto peptide derivatives and analogues of somatostatin labeled withrhenium-186 (¹⁸⁶ Re) and rhenium-188 (¹⁸⁸ Re), and methods and kits formaking, radiolabeling and using such peptides therapeutically in amammalian body.

2. Description of the Prior Art

Somatostatin is a tetradecapeptide that is endogenously produced by thehypothalamus and pancreas in humans and other mammals. The peptide hasthe formula: ##STR1## Single letter abbreviations for amino acids can befound in G. Zubay, Biochemistry (2d ed.), 1988, (MacMillan Publishing:New York), p.33!. This peptide exerts a wide variety of biologicaleffects in vivo. It is known to act physiologically on the centralnervous system, the hypothalamus, the pancreas, and the gastrointestinaltract.

Somatostatin inhibits the release of insulin and glucagon from thepancreas, inhibits growth hormone release from the hypothalamus, andreduces gastric secretions. Thus, somatostatin has clinical andtherapeutic applications for the alleviation of a number of ailments anddiseases, both in humans and other animals. Native somatostatin is oflimited utility, however, due to its short half-life in vivo, where itis rapidly degraded by peptidases. For this reason, somatostatinanalogues having improved in vivo stability have been developed in theprior art.

Freidinger, U.S. Pat. No. 4,235,886 disclose cyclic hexapeptidesomatostatin analogues useful in the treatment of a number of diseasesin humans.

Freidinger, U.S. Pat. No. 4,611,054 disclose cyclic hexapeptidesomatostatin analogues useful in the treatment of a number of diseasesin humans.

Nutt, U.S. Pat. No. 4,612,366 disclose cyclic hexapeptide somatostatinanalogues useful in the treatment of a number of diseases in humans.

Coy et al., U.S. Pat. No. 4,853,371 disclose synthetic octapeptidesomatostatin analogues.

Coy and Murphy, U.S. Pat. No. 4,871,717 disclose synthetic heptapeptidesomatostatin analogues.

Coy and Murphy, U.S. Pat. No. 4,485,101 disclose synthetic dodecapeptidesomatostatin analogues.

Coy et al., U.S. Pat. No. 4,904,642 disclose synthetic octapeptidesomatostatin analogues.

Brady, European Patent Application No. 83111747.8 discloses dicyclichexapeptide somatostatin analogues useful in the treatment of a numberof human diseases.

Bauer et al., European Patent Application No. 85810617.2 disclosesomatostatin derivatives useful in the treatment of a number of humandiseases.

Eck and Moreau, European Patent Application No. 90302760.5 disclosetherapeutic octapeptide somatostatin analogues.

Somatostatin exerts it effects by binding to specific receptorsexpressed at the cell surface of cells comprising the central nervoussystem, the hypothalamus, the pancreas, and the gastrointestinal tract.These high-affinity somatostatin binding sites have been found to beabundantly expressed at the cell surface of most endocrine-active tumorsarising from these tissues. Expression of high-affinity binding sitesfor somatostatin is a marker for these tumor cells, and specific bindingwith somatostatin can be exploited to locate and identify tumor cells invivo.

Methods for radiolabeling somatostatin analogues that have been modifiedso as to contain a tyrosine amino acid (Tyr or Y) are known in the priorart.

Albert et al., UK Patent Application 8927255.3 disclose radioimagingusing somatostatin derivatives such as octreotide labeled with ¹²³ I.

Bakker et al., J. Nucl. Med. 31: 1501-1509 (1990) describe radioactiveiodination of a somatostatin analog and its usefulness in detectingtumors in vivo.

Bakker et al., J. Nucl. Med. 32: 1184-1189 (1991) teach the usefulnessof radiolabeled somatostatin for radioimaging in vivo.

Alternatively, methods for radiolabeling somatostatin by covalentlymodifying the peptide to contain a radionuclide-chelating group havebeen disclosed in the prior art.

Albert et al., UK Patent Application 8927255.3 disclose radioimagingusing somatostatin derivatives such as octreotide labeled with ¹¹¹ Invia a chelating group bound to the amino-terminus.

Albert et al., European Patent Application No. WO 91/01144 discloseradioimaging using radiolabeled peptides related to growth factors,hormones, interferons and cytokines and comprised of a specificrecognition peptide covalently linked to a radionuclide chelating group.

Kwekkeboom et al., J. Nucl. Med. 32: 981 (1991) Abstract #305 relates toradiolabeling somatostatin analogues with ¹¹¹ In.

Albert et al., Abstract LM10, 12th American Peptide Symposium: 1991describe uses for ¹¹¹ In-labeled diethylene-triaminopentaaceticacid-derivatized somatostatin analogues.

These methods can be readily adapted to enable detection of tumor cellsin vivo based on their expression of high affinity binding sites forsomatostatin by radioimaging. Radionuclides which emit high energy gammaradiation can be readily detected by scintigraphy after injection into ahuman or an animal. A variety of radionuclides are known to be usefulfor radioimaging, including ⁶⁷ Ga, ^(99m) Tc (Tc-99m), ¹¹¹ In, ¹²³ I,¹²⁵ I, ¹⁶⁹ Yb or ¹⁸⁶ Re. The sensitivity of imaging methods usingradioactively-labeled peptides is much higher than other techniquesknown in the art, since the specific binding of the radioactive peptideconcentrates the radioactive signal over the cells of interest, forexample, tumor cells. This is particularly important forendocrine-active gastrointestinal tumors, which are usually small,slow-growing and difficult to detect by conventional methods. Labelingwith technetium-99m (Tc-99m) is advantageous because the nuclear andradioactive properties of this isotope make it an ideal scintigraphicimaging agent. Tc-99m has a single photon energy of 140 keV and aradioactive half-life of about 6 hours, and is readily available from a⁹⁹ Mo-^(99m) Tc generator. Other radionuclides have effective half-liveswhich are much longer (for example, ¹¹¹ In, which has a half-life of60-70 h) or are toxic (for example, ¹²⁵ I). Although Tc-99m is an idealradiolabeling reagent, it has not been widely used in the art prior tothe present invention see, for example, Lamberts, J. Nucl. Med. 32:1189-1191 (1991)!.

Radiolabeled somatostatin analogues can also be used therapeutically.For these applications, the rhenium isotopes ¹⁸⁶ Re and ¹⁸⁸ Re areparticularly advantageous.

The use of chelating agents for radiolabeling polypeptides, and methodsfor labeling peptides and polypeptides with Tc-99m are known in theprior art and are disclosed in copending U.S. patent applications Ser.Nos. 07/653,012, now abandoned, which has been allowed as U.S. Ser. No.08/480,551; 07/807,062, now U.S. Pat. No. 5,443,815; 07/851,074, nowabandoned, which issued as U.S. Pat. No. 5,711,931; 07/871,282, whichissued as U.S. Pat. No. 5,720,934; and 07/886,752, now abandoned, whichhas been allowed as U.S. Ser. No. 08/273,274 which are herebyincorporated by reference.

Fritzberg, U.S. Pat. No. 4,444,690 describes a series oftechnetium-chelating agents based on 2,3-bis(mercaptoacetamido)propanoate.

Gansow et al., U.S. Pat. No. 4,472,509 teach methods of manufacturingand purifying Tc-99m chelate-conjugated monoclonal antibodies.

Reno and Bottino, European Patent Application 87300426.1 discloseradiolabeling antibodies with Tc-99m.

Pak et al., European Patent Application No. WO 88/07382 disclose amethod for labeling antibodies with Tc-99m.

Rhodes, Sem. Nucl. Med. 4: 281-293 (1974) teach the labeling of humanserum albumin with technetium-99m.

Khaw et al., J. Nucl. Med. 23: 1011-1019 (1982) disclose methods forlabeling biologically active macromolecules with Tc-99m.

Byrne and Tolman, supra, disclose a bifunctional thiolactone chelatingagent for coupling Tc-99m to biological molecules.

Cox et al., Abstract, 7th International Symposium on Radiopharmacology,p. 16, 1991, disclose the use of, Tc-99m-, ¹³¹ I- and ¹¹¹ In-labeledsomatostatin analogues in radiolocalization of endocrine tumors in vivoby scintigraphy.

Methods for directly labeling somatostatin, derivatives of somatostatin,analogues of somatostatin or peptides that bind to the somatostatinreceptor and contain at least 2 cysteine residues that form a disulfideor wherein the disulfide is reduced to the sulfhydryl form, aredisclosed in co-pending U.S. patent application Ser. No. 07/807,062,which is hereby incorporated by reference.

There remains a need for synthetic (to make routine manufacturepracticable and to ease regulatory acceptance) somatostatin analogueshaving increased in vivo stability, to be used therapeutically and asscintigraphic agents when radiolabeled with Tc-99m for use in imagingtumors in vivo. Small synthetic somatostatin analogues are provided bythis invention that specifically fulfill this need.

SUMMARY OF THE INVENTION

The present invention provides somatostatin analogues for therapeuticand scintigraphic imaging applications that are peptide reagents.Specifically, the invention provides peptide reagents for preparingscintigraphic imaging agents that are technetium-99m (Tc-99m) labeled.The scintigraphic imaging agents of the invention are comprised of apeptide that is a somatostatin analogue covalently linked to a Tc-99mbinding moiety and labeled with Tc-99m. In addition, the inventionprovides somatostatin analogues and such analogues radiolabeled with ¹⁸⁶Re and ¹⁸⁸ Re that are useful therapeutically.

The somatostatin analogues provided by the invention aresomatostatin-receptor binding peptides having the following formula:##STR2## where R¹ and R² are independently H, lower alkyl or substitutedalkyl, aryl or substituted aryl;

R³ and R⁴ are each independently H, lower alkyl or substituted alkyl,aryl or substituted aryl, or either R³ or R⁴ are N(R¹⁰)₂, where each R¹⁰is independently H, lower alkyl or a peptide sequence of no more than 10amino acids, and m is an integer between 0 and 3;

X¹ and X² are each independently a D- or L- amino acid, and n and q areindependently either 0 or 1;

A¹ is D- or L-Phe or D- or L-Tyr or substituted derivatives thereof;

A² is D-or L- Trp or substituted derivatives thereof;

A³ is D- or L-Lys or substituted derivatives thereof;

A⁴ is Thr, Ser, Val, Phe, Ile or 2-amino-isobutyric acid (Aib), mostpreferably Thr or Val;

X³ is H, --COOR⁹, --CH₂ OH, CH₂ COOR⁹, or --CON(R⁹)₂, where each R⁹ isindependently H, lower linear or cyclic alkyl or substituted derivativesthereof, or a peptide having an amino acid sequence of no more than 10residues;

R⁵ and R⁶ are each independently H or lower alkyl and p is either 0, 1or 2; and

R⁷ and R⁸ are independently H, lower alkyl or substituted lower alkyl,or either R⁷ and R⁸ are --COOH or a derivative thereof.

In a preferred embodiment, A¹ is Phe or Tyr, A² is Trp or mostpreferably D-Trp, A³ is Lys and A⁴ is Thr or Val.

In a first aspect of the present invention are provided peptide reagentsthat are somatostatin analogues as described herein having increased invivo stability compared with native somatostatin, and that aretherapeutically useful in the alleviation of diseases or other ailmentsin humans or other animals.

The invention also provides pharmaceutical compositions comprising thesomatostatin receptor-binding peptides of the invention in apharmaceutically acceptable carrier.

The invention also provides a method for alleviatingsomatostatin-related diseases in animals, preferably humans, comprisingadministering a therapeutically effective amount of the somatostatinanalogues of the invention to the animal. In preferred embodiments, theamount of the somatostatin analogue administered is from about 0.1 toabout 50 mg/kg body weight/day.

Another aspect of the present invention provides reagents for preparingscintigraphic imaging agents, each reagent comprising a peptide that isa somatostatin analogue and is covalently linked to a Tc-99m bindingmoiety.

A first aspect of the reagents for preparing scintigraphic imagingagents of the invention are reagents, each comprised of a peptide thatis a somatostatin analogue that is covalently linked to a Tc-99m bindingmoiety having formula:

    C(pgp).sup.s -(aa)-C(pgp).sup.s

wherein C(pgp)^(s) is a protected cysteine and (aa) is an amino acid. Ina preferred embodiment, the amino acid is glycine.

In a second embodiment, the invention provides peptide reagents capableof being Tc-99m labeled for imaging sites within a mammalian body, eachcomprising a somatostatin analogue that is covalently linked to aradioisotope complexing group comprising a single thiol moiety havingthe following structure:

    A-CZ(B)- C(RR')!.sub.n -X

wherein

A is H, HOOC, H₂ NOC, or --NHOC;

B is SH or NHR";

X is H, methyl, SH or NHR";

Z is H or methyl;

R and R' are independently H or lower alkyl;

R" is H, lower alkyl or --C═O;

n is 0, 1 or 2;

and where B is NHR", X is SH, Z is H and n is 1 or 2; where X is NHR", Bis SH, Z is H and n is 1 or 2; where B is H, A is HOOC, H₂ NOC, or--NHOC, X is SH, Z is H and n is 0 or 1; where Z is methyl, X is methyl,A is HOOC, H₂ NOC, or --NHOC,, B is SH and n is 0; and wherein the thiolmoiety is in the reduced form. In a preferred embodiment, the peptide iscomprised between 4 and 30 amino acids.

In another embodiment, the invention provides peptide reagents capableof being labeled with Tc-99m for imaging sites within a mammalian body,each comprising a somatostatin analogue that is covalently linked to aTc-99m binding moiety of formula: ##STR3## for purposes of thisinvention, radiolabel-binding moieties having this structure will bereferred to as picolinic acid (Pic)-based moieties! or ##STR4## whereinX is H or a protecting group; (amino acid) is any amino acid and theradiolabel-binding moiety is covalently linked to the peptide. Forpurposes of this invention, radiolabel-binding moieties having thisstructure will be referred to as picolylamine (Pica)-based moieties. Ina preferred embodiment, the amino acid is glycine and X is anacetamidomethyl protecting group. In additional preferred embodiments,the peptide is comprised between 4 and 30 amino acids.

Yet another embodiment of the invention provides peptide reagentscapable of being labeled with Tc-99m for imaging sites within amammalian body, each comprising a somatostatin analogue that iscovalently linked to a Tc-99m binding moiety that is a bisamino bisthiolTc-99m binding moiety. The bisamino bisthiol Tc-99m binding moiety inthis embodiment of the invention has a formula selected from the groupconsisting of: ##STR5## wherein each R can be independently H, CH₃ or C₂H₅ ;

each (pgp)^(s) can be independently a thiol protecting group or H;

m, n and p are independently 2 or 3;

A is linear or cyclic lower alkyl, aryl, heterocyclyl, combinations orsubstituted derivatives thereof; and

X is peptide; and ##STR6## wherein each R is independently H, CH₃ or C₂H₅ ;

m, n and p are independently 2 or 3;

A is linear or cyclic lower alkyl, aryl, heterocyclyl, combinations orsubstituted derivatives thereof;

V is H or CO-peptide;

R' is H or peptide;

provided that when V is H, R' is peptide and when R' is H, V isCO-peptide. For purposes of this invention, radiolabel-binding moietieshaving these structures will be referred to as "BAT" moieties. In apreferred embodiment, the peptide is comprised between 4 and 30 aminoacids.

The invention also comprises scintigraphic imaging agents that arecomplexes of the peptide reagents of the invention with Tc-99m andmethods for radiolabeling the peptide reagents of the invention withTc-99m. Radiolabeled complexes provided by the invention are formed byreacting the peptide reagents of the invention with Tc-99m in thepresence of a reducing agent. Preferred reducing agents include but arenot limited to dithionite ion, stannous ion and ferrous ion. Complexesof the invention are also formed by labeling the peptide reagents of theinvention with Tc-99m by ligand exchange of a prereduced Tc-99m complexas provided herein.

The invention also provides kits for preparing scintigraphic imagingagents that are the peptide reagents of the invention radiolabeled withTc-99m. Kits for labeling the peptide reagents of the invention withTc-99m are comprised of a sealed vial containing a predeterminedquantity of a peptide reagent of the invention and a sufficient amountof reducing agent to label the peptide with Tc-99m.

This invention provides methods for preparing peptide reagents of theinvention by chemical synthesis in vitro. In a preferred embodiment,peptides are synthesized by solid phase peptide synthesis.

This invention provides methods for using scintigraphic imaging agentsthat are Tc-99m labeled peptide reagents for imaging sites within amammalian body by obtaining in vivo gamma scintigraphic images. Thesemethods comprise administering an effective diagnostic amount of Tc-99mlabeled peptide reagents of the invention and detecting the gammaradiation emitted by the Tc-99m label localized at the site within themammalian body.

This invention provides reagents for preparing a radiolabledsomatostatin receptor-binding agent comprising the somatostatinreceptor-binding peptides of the invention covalently linked to aradiolabel-binding moiety. In a preferred embodiment, the reagent isradioactively labeled with Tc-99m. In another preferred embodiment, thereagent is radioactively labeled with ¹⁸⁶ Re or ¹⁸⁸ Re.

The invention also provides methods for alleviating somatostatin-relateddiseases in animals, preferably humans, comprising administering atherapeutically effective amount of the radiolabeledsomatostatin-binding peptide reagents of the invention to the animal. Inpreferred embodiments, the reagent is radioactively labeled with ¹⁸⁶ Reor ¹⁸⁸ Re.

Specific preferred embodiments of the present invention will becomeevident from the following more detailed description of certainpreferred embodiments and the claims.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides peptide reagents for preparingradiolabeled imaging agents for imaging site within a mammalian body.The peptide reagents of the invention each comprise a somatostatinanalogue that is covalently linked to a Tc-99m binding moiety. Theinvention also provides somatostatin analogues having an increased invivo stability and that are useful for alleviating diseases or otherailments in humans or other animals.

The invention provides a method for using the somatostatin analogues ofthe invention to alleviate diseases or other ailments in animals,preferably humans. These diseases and ailments include but are notlimited to diabetes and diabetes-related retinopathy, cirrhosis of theliver and hepatitis infection, bleeding ulcers and othergastrointestinal bleeding, pancreatitis, central nervous systemdisorders, endocrine disorders, Alzheimer's disease, acromegaly andother diseases and disorders related to the production of inappropriatelevels of growth hormone in vivo, and cancer, particularly those cancerswhose growth is dependent or influenced by growth hormone production.Dosages of the somatostatin analogues provided by the invention may bethe same as those dosages of native somatostatin routinely used fortreatment of the above or other diseases, or less of the compounds ofthe invention may be administered due to their longer in vivo half-life.

Labeling with Tc-99m is an advantage of the present invention becausethe nuclear and radioactive properties of this isotope make it an idealscintigraphic imaging agent. This isotope has a single photon energy of140 keV and a radioactive half-life of about 6 hours, and is readilyavailable from a ⁹⁹ Mo-^(99m) Tc generator. Other radionuclides known inthe prior art have effective half-lives which are much longer (forexample, ¹¹¹ In, which has a half-life of 67.4 h) or are toxic (forexample, ¹²⁵ I).

Radiotherapeutic embodiments of the invention, on the other hand, areadvantageously labeled with ¹⁸⁶ Re or ¹⁸⁸ Re. Such embodiments areuseful in the treatment of somatostatin-related diseases or otherailments in animals, preferably humans, including but not limited tocancer and other diseases characterized by the growth of malignant orbenign tumors capable of binding somatostatin or somatostatin analoguesvia the expression of somatostatin receptors on the cell surface ofcells comprising such tumors.

In the Tc-99m binding moieties and peptides covalently linked to suchmoieties that contain a thiol covalently linked to a thiol protectinggroups (pgp)^(s) ! provided by the invention, the thiol-protectinggroups may be the same or different and may be but are not limited to:

--CH₂ -aryl (aryl is phenyl or alkyl or alkyloxy substituted phenyl);

--CH-(aryl)₂, (aryl is phenyl or alkyl or alkyloxy substituted phenyl);

--C-(aryl)₃, (aryl is phenyl or alkyl or alkyloxy substituted phenyl);

--CH₂ -(4-methoxyphenyl);

--CH-(4-pyridyl)(phenyl)₂ ;

--C(CH₃)₃

-9-phenylfluorenyl;

--CH₂ NHCOR (R is unsubstituted or substituted alkyl or aryl);

--CH₂ --NHCOOR (R is unsubstituted or substituted alkyl or aryl);

--CONHR (R is unsubstituted or substituted alkyl or aryl);

--CH₂ --S--CH₂ -phenyl

Preferred protecting groups have the formula --CH₂ --NHCOR wherein R isa lower alkyl having 1 and 8 carbon atoms, phenyl or phenyl-substitutedwith lower alkyl, hydroxyl, lower alkoxy, carboxy, or loweralkoxycarbonyl. The most preferred protecting group is anacetamidomethyl group.

Each specific-binding peptide provided by the invention is comprised ofa sequence of amino acids. The term amino acid as used in this inventionis intended to include all L- and D- amino acids, naturally occurringand otherwise, and substituted derivatives thereof. Specific-bindingpeptides provided by the invention include but are not limited topeptides having the following sequences: ##STR7##

Specific-binding peptides of the present invention can be chemicallysynthesized in vitro. Peptides of the present invention can generallyadvantageously be prepared on an amino acid synthesizer. The peptides ofthis invention can be synthesized wherein the radiolabel-binding moietyis covalently linked to the peptide during chemical synthesis in vitro,using techniques well known to those with skill in the art. Suchpeptides covalently-linked to the radiolabel-binding moiety duringsynthesis are advantageous because specific sites of covalent linkagecan be determined.

Radiolabel binding moieties of the invention may be introduced into thetarget specific peptide during peptide synthesis. For embodimentscomprising picolinic acid (Pic-); e.g., Pic-Gly-Cys(protecting group)-!,the radiolabel-binding moiety can be synthesized as the last (i.e.,amino-terminal) residue in the synthesis. In addition, the picolinicacid-containing radiolabel-binding moiety may be covalently linked tothe ε-amino group of lysine to give, for example, αN(Fmoc)-Lys-εNPic-Gly-Cys(protecting group)!, which may be incorporated at anyposition in the peptide chain. This sequence is particularlyadvantageous as it affords an easy mode of incorporation into the targetbinding peptide.

Similarly, the picolylamine (Pica)-containing radiolabel-binding moiety-Cys(protecting group)-Gly-Pica! can be prepared during peptidesynthesis by including the sequence -Cys(protecting group)-Gly-! at thecarboxyl terminus of the peptide chain. Following cleavage of thepeptide from the resin the carboxyl terminus of the peptide is activatedand coupled to picolylamine. This synthetic route requires that reactiveside-chain functionalities remain masked (protected) and do not reactduring the conjugation of the picolylamine.

This invention also provides small synthetic peptides that aresomatostatin analogues and incorporate bisamine bisthiol (BAT) chelatorsthat may be labeled with Tc-99m.

This invention provides for the incorporation of these chelators intovirtually any position in the peptide, via covalent linkage to anyappropriate functional group of the peptide, except that the chelatingmoieties of the invention are not covalently linked to functional groupscomprising the amino acid side chains of the amino acids A¹, A², A³ orA⁴.

In forming a complex of radioactive technetium with the reagents of thisinvention, the technetium complex, preferably a salt of Tc-99mpertechnetate, is reacted with the reagent in the presence of a reducingagent. Preferred reducing agents are dithionite, stannous and ferrousions; the most preferred reducing agent is stannous chloride. Means forpreparing such complexes are conveniently provided in a kit formcomprising a sealed vial containing a predetermined quantity of areagent of the invention to be labeled and a sufficient amount ofreducing agent to label the reagent with Tc-99m. Alternatively, thecomplex may be formed by reacting a reagent of this invention with apre-formed labile complex of technetium and another compound known as atransfer ligand. This process is known as ligand exchange and is wellknown to those skilled in the art. The labile complex may be formedusing such transfer ligands as tartrate, citrate, gluconate or mannitol,for example. Among the Tc-99m pertechnetate salts useful with thepresent invention are included the alkali metal salts such as the sodiumsalt, or ammonium salts or lower alkyl ammonium salts.

It is an advantage of the somatostatin analogues provided by thisinvention that the thioether linkage is stable under the conditions ofTc-99m conjugation to the covalently linked Tc-99m binding moiety. Incontrast, Tc-99m conjugation to a Tc-99m binding moiety covalentlylinked to native somatostatin, or to a somatostatin analogue having adisulfide bond, can result in reduction of the disulfide accompanied bya loss of biological activity. Such loss of biological activity can alsooccur in vivo using native somatostatin, or to any somatostatin analoguehaving a disulfide bond. The present invention is not subject to similarlosses in biological activity in vivo because the thioether linkage ineach of the somatostatin analogues of the invention is a stable covalentbond.

It is another advantage of the somatostatin analogues provided by thisinvention that the covalent linkage between the amino terminus and thecysteine protecting moiety acts to protect the peptide from degradationby exopepetidases.

In a preferred embodiment of the invention, a kit for preparingtechnetium-labeled peptides is provided. An appropriate amount of thepeptide reagent is introduced into a vial containing a reducing agent,such as stannous chloride, in an amount sufficient to label the peptidewith Tc-99m. An appropriate amount of a transfer ligand as described(such as tartrate, citrate, gluconate or mannitol, for example) can alsobe included. The kit may also contain conventional pharmaceuticaladjunct materials such as, for example, pharmaceutically acceptablesalts to adjust the osmotic pressure, buffers, preservatives and thelike. The components of the kit may be in liquid, frozen or dry form. Ina preferred embodiment, kit components are provided in lyophilized form.

Radiolabeled imaging reagents according to the present invention may beprepared by the addition of an appropriate amount of Tc-99m or Tc-99mcomplex into the vials and reaction under conditions described inExample 2 hereinbelow.

Radioactively-labeled scintigraphic imaging agents provided by thepresent invention are provided having a suitable amount ofradioactivity. In forming Tc-99m radioactive complexes, it is generallypreferred to form radioactive complexes in solutions containingradioactivity at concentrations of from about 0.01 millicurie (mCi) to100 mCi per mL.

The imaging reagents provided by the present invention can be used forvisualizing organs such as the kidney for diagnosing disorders in theseorgans, and tumors, in particular gastrointestinal tumors, myelomas,small cell lung carcinoma and other APUDomas, endocrine tumors such asmedullary thyroid carcinomas and pituitary tumors, brain tumors such asmeningiomas and astrocytomas, and tumors of the prostate, breast, colon,and ovaries can also be imaged. In accordance with this invention, theTc-99m labeled peptide reagents are administered in a single unitinjectable dose. The Tc-99m labeled peptide reagents provided by theinvention may be administered intravenously in any conventional mediumfor intravenous injection such as an aqueous saline medium, or in bloodplasma medium. Generally, the unit dose to be administered has aradioactivity of about 0.01 mCi to about 100 mCi, preferably 1 mCi to 20mCi. The solution to be injected at unit dosage is from about 0.01 mL toabout 10 mL. After intravenous administration, imaging in vivo can takeplace in a matter of a few minutes. However, imaging can take place, ifdesired, in hours or even longer, after the radiolabeled peptide isinjected into a patient. In most instances, a sufficient amount of theadministered dose will accumulate in the area to be imaged within about0.1 of an hour to permit the taking of scintiphotos. Any conventionalmethod of scintigraphic imaging for diagnostic purposes can be utilizedin accordance with this invention.

The methods for making and labeling these compounds are more fullyillustrated in the following Examples. These Examples illustrate certainaspects of the above-described method and advantageous results, and areshown by way of illustration and not limitation.

EXAMPLE 1 Solid Phase Peptide Synthesis

Solid phase peptide synthesis (SPPS) was carried out on a 0.25 millimole(mmole) scale using an Applied Biosystems Model 431A Peptide Synthesizerand using 9-fluorenylmethyloxycarbonyl (Fmoc) amino-terminus protection,coupling with dicyclohexylcarbodiimide/hydroxybenzotriazole or2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluroniumhexafluorophosphate/hydroxybenzotriazole (HBTU/HOBT), and usingp-hydroxymethylphenoxymethyl-polystyrene (HMP) resin forcarboxyl-terminus acids or Rink amide resin for carboxyl-terminusamides. Resin-bound products were routinely cleaved using a solutioncomprised of trifluoroacetic acid, water, thioanisole, ethanedithiol,and triethylsilane, prepared in ratios of 100:5:5:2.5:2 for 1.5-3 h atroom temperature.

2-chloro- or 2-bromoacetyl groups were introduced either by using theappropriate 2-haloacetic acid as the last residue coupled during SPPS,or by treating the N-terminus free amino acid peptide bound to the resinwith either 2-haloaceticacid/diisopropylcarbodiimide/N-hydroxysuccinimide/NMP or 2-haloaceticanhydride/diisopropylethylamine/NMP.

HPLC-purified 2-haloacetylated peptides were cyclized by stirring an0.1-1.0 mg/mL solution in phosphate or bicarbonate buffer (pH 8.0)containing 0.5-1.0 mM EDTA for 4-48 h followed by acidification withacetic acid, lyophilization and HPLC purification. Crude peptides werepurified by preparative high pressure liquid chromatography (HPLC) usinga Waters Delta Pak C18 column and gradient elution using 0.1%trifluoroacetic acid (TFA) in water modified with acetonitrile.Acetonitrile was evaporated from the eluted fractions which were thenlyophilized. The identity of each product was confirmed by fast atombombardment mass spectroscopy (FABMS).

The following somatostatin analogues were synthesized as providedherein, and the products of such synthesis identified by FABMS (MH⁺values in parentheses): ##STR8##

EXAMPLE 2 A General Method for Radiolabeling with Tc-99m

0.1 mg of a peptide prepared as in Example 2 was dissolved in 0.1 mL ofwater or 50 mM potassium phosphate buffer (pH=5, 6 or 7.4). Tc-99mgluceptate was prepared by reconstituting a Glucoscan vial (E.I. DuPontde Nemours, Inc.) with 1.0 mL of Tc-99m sodium pertechnetate containingup to 200 mCi and allowed to stand for 15 minutes at room temperature.25 μl of Tc-99m gluceptate was then added to the peptide and thereaction allowed to proceed at room temperature or at 100° C. for 15-30min and then filtered through a 0.2 μm filter.

The Tc-99m labeled peptide purity was determined by HPLC using thefollowing conditions:

1. The peptide is dissolved in 50 mM potassium phosphate buffer (pH 7.4)and labeled at room temperature.

2. The peptide is dissolved in 50 mM potassium phosphate buffer (pH 7.4)and labeled at 100° C.

3. The peptide is dissolved in water and labeled at room temperature.

4. The peptide is dissolved in water and labeled at 100° C.

5. The peptide is dissolved in 50 mM potassium phosphate buffer (pH 6.0)and labeled at 100° C.

6. The peptide is dissolved in 50 mM potassium phosphate buffer (pH 5.0)and labeled at room temperature.

General HPLC methods are as follows:

Solvents

solvent A=0.1% CF₃ COOH/H₂ O

solvent B₇₀ =0.1% CF₃ COOH/70% CH₃ CN/H₂ O

solvent B₉₀ =0.1% CF₃ COOH/90% CH₃ CN/H₂ O

solvent flow rate=1 mL/min

Columns

Vydak 218TP54 RP-18, 5μ×220 mm×4.6 mm analytical column with guardcolumn

Brownlee Spheri-5 RP-18, 5μ×220 mm×4.6 mm column

Methods

1. Brownlee column 100% A to 100% B₇₀ in 10 min

2. Vydak column 100% A to 100% B₉₀ in 10 min

3. Vydak column 100% A to 100% B₇₀ in 10 min

Radioactive components were detected by an in-line radiometric detectorlinked to an integrating recorder. Tc-99m gluceptate and Tc-99m sodiumpertechnetate elute between 1 and 4 minutes under these conditions,whereas the Tc-99m labeled peptide eluted after a much greater amount oftime.

It should be understood that the foregoing disclosure emphasizes certainspecific embodiments of the invention and that all modifications oralternatives equivalent thereto are within the spirit and scope of theinvention as set forth in the appended claims.

    __________________________________________________________________________    SEQUENCE LISTING                                                              (1) GENERAL INFORMATION:                                                      (iii) NUMBER OF SEQUENCES: 2                                                  (2) INFORMATION FOR SEQ ID NO:1:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 5 amino acids                                                     (B) TYPE: amino acid                                                          (D) TOPOLOGY: circular                                                        (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Modified-site                                                   (B) LOCATION: 1..5                                                            (D) OTHER INFORMATION: /label=Cyclized                                        /note= "The peptide is cyclized between the                                   sidechain sulfur of the cysteine residue and the                              amino terminus via an acetamido group; the trp                                (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                       TyrTrpLysThrCys                                                               15                                                                            (2) INFORMATION FOR SEQ ID NO:2:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 6 amino acids                                                     (B) TYPE: amino acid                                                          (D) TOPOLOGY: circular                                                        (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Modified-site                                                   (B) LOCATION: 1..5                                                            (D) OTHER INFORMATION: /label=Cyclized                                        /note= "The peptide is cyclized between the                                   sidechain sulfur of the cysteine residue and the                              amino terminus via an acetamido group; the trp                                (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                       TyrTrpLysThrCysThr                                                            15                                                                            __________________________________________________________________________

What is claimed is:
 1. A reagent for preparing a scintigraphic imagingagent comprising a peptide having a formula: ##STR9## wherein R¹ and R²are independently H, lower alkyl or substituted alkyl, aryl orsubstituted aryl; R³ and R⁴ are each independently H, lower alkyl orsubstituted alkyl, aryl or substituted aryl, or either R³ or R⁴ areN(R¹⁰)₂, where each R¹⁰ is independently H, lower alkyl or a peptidesequence of no more than 10 amino acids, and m is an integer between 0and 3; X¹ and x² are each independently a D- or L- amino acid, and n andq are independently either 0 or 1; A¹ is D- or L-Phe or D- or L-Tyr or asubstituted derivative thereof; A² is D- or L-Trp or a substitutedderivative thereof; A³ is D- or L-Lys or a substituted derivatives!derivative thereof; A⁴ is Thr, Ser, Val, Phe, Ile or Aib; X³ is --COOR⁹,--CH₂ OH, CH₂ COOR⁹, or --CON⁹)₂, where each R⁹ is independently H,lower linear or cyclic alkyl or a substituted derivative thereof, or apeptide having an amino acid sequence of no more than 10 residues; R⁵and R⁶ are each independently H or lower alkyl and p is either 0, 1 or2; and R⁷ and R⁸ are independently H, lower alkyl or substituted loweralkyl, or either R⁷ or R⁸ are --COOH or a derivative thereof; whereinsaid peptide binds somatostatin receptor; a carboxyl terminus of saidpeptide being covalently linked to a technetium-99m binding moietyselected from the group consisting of:

    C(pgp).sup.s -(aa)-C(pgp).sup.s

wherein C(pgp)^(s) is a cysteine having a protected thiol group and (aa)is any primary α or β-amino acid; a technetium-99m binding moietycomprising a single-thiol moiety having a formula:

    A-CZ(B)- C(RR')!.sub.n -X

wherein A is H, HOOC, H₂ NOC, or --NHOC; B is SH or NHR"; X is H,methyl, SH or NHR"; Z is H or methyl; R and R' are independently H orlower alkyl; R" is H, lower alkyl or --C═O; n is 0, 1 or2; and where Bis NHR", X is SH, Z is H and n is 1 or 2; where X is NHR", B is SH, Z isH and n is l or 2; here B is H, A is HOOC, H₂ NOC, or --NHOC, X is SH, Zis H and n is 0 or 1; where Z is methyl, X is methyl, A is HOOC, H₂ NOC,or --NHOC, B is SH and n is 0; and wherein the thiol moiety is in thereduced form; ##STR10## wherein X=H or a protecting group; (aminoacid)=any amino acid; ##STR11## wherein X=H or a protecting group;(amino acid)=any amino acid; ##STR12## wherein each R is independentlyH, CH₃ or C₂ H₅ ; each (pgp)^(s) is independently a thiol protectinggroup or H; m, n and p are independently 2 or 3; A=linear or cycliclower alkyl, aryl, heterocyclyl, a combination thereof or a substitutedderivative thereof; and ##STR13## wherein each R is independently H, CH₃or C₂ H₅ ; m, n and p are independently 2 or 3; A=linear or cyclic loweralkyl, aryl, heterocyclyl, a combination thereof or a substitutedderivative thereof, V=H or --CO-peptide; R'=H or peptide; and whereinwhen V=H, R'=peptide and when R'=H, V=--CO-peptide and wherein thetechnetium-99m binding moiety is capable of forming an electricallyneutral complex with technetium-99m.
 2. The reagent of claim 1 whereinthe technetium-99m binding moiety is C(pgp)^(s) -(aa)-C(pgp)^(s) and thethiol protecting group has a formula

    --CH.sub.2 --NH--CO--R

wherein R is a lower alkyl having 1 to 6 carbon atoms, a 2-pyridyl, a3-pyridyl, a 4-pyridyl, a phenyl, or a phenyl substituted with a loweralkyl, a hydroxy, a lower alkoxy, a carboxy, or a lower alkoxycarbonyl.3. The reagent of claim 1 wherein the technetium-99m binding moiety hasa formula: ##STR14##
 4. The reagent according to claim 1, wherein thepeptide is chemically synthesized in vitro.
 5. The reagent according toclaim 4, wherein the peptide is synthesized by solid phase peptidesynthesis.
 6. The reagent according to claim 4, wherein thetechnetium-99m binding moiety is covalently linked to the peptide duringin vitro chemical synthesis.
 7. The reagent according to claim 6,wherein the technetium-99m binding moiety is covalently linked to thepeptide during solid phase peptide synthesis.
 8. A compositioncomprising a reagent comprising a somatostatin receptor-binding peptideselected from the group consisting of: ##STR15## said peptide beingcovalently linked to a technetium-99m binding moiety selected from thegroup consisting of:

    C(pgp).sup.s -(aa)-C(pgp).sup.s

wherein C(pgp)^(s) is a cysteine having a protected thiol group and (aa)is any primary α- or β- amino acid; a technetium-99m binding moietycomprising a single-thiol moiety having a formula:

    A-CZ(B)- C(RR')!.sub.n -X

wherein A is H, HOOC, H₂ NOC, or --NHOC; B is SH or NHR"; X is H, methl,SH or NHR"; Z is H or methyl; R and R' are independently H or loweralkyl, R" is H, lower alkyl or --C═O; n is 0, 1 or2; and where B isNHR", X is SH, Z is H and n is 1 or 2; where X is NHR", B is SH, Z is Hand n is 1 or 2; where B is H, A is HOOC, H₂ NOC, or --NHOC, X is SH, Zis H and n is 0 or 1; where Z is methyl, X is methyl, A is HOOC, H₂ NOC,or --NHOC, B is SH and n is 0; and wherein the thiol moiety is in thereduced form; ##STR16## wherein X=H or a protecting group; (aminoacid)=any amino acid; ##STR17## wherein X=H or a protecting group;(amino acid)=any amino acid; ##STR18## wherein each R is independentlyH, CH₃ or C₂ H₅ ; each (pgp)^(s) is independently a thiol protectinggroup or H: m, n and p are independently 2 or 3; A=linear or cycliclower alkyl, aryl, heterocyclyl, a combination thereof or a substitutedderivative thereof; and ##STR19## wherein each R is independently H, CH₃or C₂ H₅ ; m, n and p are independently 2 or 3; A=linear or cyclic loweralkyl, aryl, heterocyclyl, a combination thereof or a substitutedderivative thereof; V=H or --CO-peptide; R'=H or peptide; and whereinwhen V=H, R'=peptide and when R'=H, V=--CO-peptide.
 9. A reagent forpreparing a radiolabled somatostatin receptor-binding agent comprising apeptide having a formula: ##STR20## wherein R¹ and R² are independentlyH, lower alkyl or substituted alkyl, aryl or substituted aryl; R³ and R⁴are each independently H, lower alkyl or substituted alkyl, aryl orsubstituted aryl, or either R³ or R⁴ are N(R¹⁰)₂, where each R¹⁰ isindependently H, lower alkyl or a peptide sequence of no more than 10amino acids, and m is an integer between 0 and 3; X¹ and X² are eachindependently a D- or L- amino acid, and n and q are independentlyeither 0 or 1; A¹ is D- or L-Phe or D- or L-Tyr or a substitutedderivative thereof; A² is D- or L-Trp or a substituted derivativethereof; A³ is D- or L-Lys or a substituted derivative thereof; A⁴ isThr, Ser, Val, Phe, Ile or Aib; X³ is --COOR⁹, --CH₂ OH, CH₂ COOR⁹, or--CON(R⁹)₂, where each R⁹ is independently H, lower linear or cyclicalkyl or a substituted derivative thereof, or a peptide having an aminoacid sequence of no more than 10 residues; R⁵ and R⁶ are eachindependently H or lower alkyl and p is either 0, 1 or 2; and R⁷ and R⁸are independently H, lower alkyl or substituted lower alkyl, or eitherR⁷ or R⁸ are --COOH or a derivative thereof; wherein said peptide bindssomatostatin peptide; and a radiolabel-binding moiety covalently linkedthereto.
 10. The reagent according to claim 9, wherein the peptide ischemically synthesized in vitro.
 11. The reagent according to claim 10,wherein the radiolabel binding moiety is covalently linked to thepeptide during solid phase peptide synthesis.